1. Field of the Invention
The invention is generally related to the measurement of the length of unknown deoxyribonucleic acid (DNA) fragments using an electrophoresis system and, more specifically, to the manufacture and use of bracketing pairs of DNA standards closely related in sequence and preferably derived from the DNA locus to be measured.
2. Definitions
U.S. Pat. No. 5,364,759 to Caskey et al. provides a concise definition for several terms used in biotechnology, particularly as they pertain to short tandem repeat polymorphisms. The complete contents of U.S. Pat. No. 5,364,759 is hereby incorporated by reference. The following definitions are provided to assist in providing a clear and consistent understanding of the invention.
Allele: a genetic variation associated with a segment of DNA, i.e., one of two or more alternate forms of a DNA sequence occupying the same polymorphic region of a genetic locus.
Allelic ladder: standard size marker consisting of amplified alleles from the polymorphic region.
Biochemical nomenclature: standard biochemical nomenclature is used herein in which the nucleotide bases are designated as adenine, A; thymine, T; guanine, G; and cytosine, C. Corresponding nucleotides are, for example, deoxyguanosine-5'triphosphate (dGTP), etc.
Deviation: the difference between the known (expected) length of a polymorphic DNA fragment and its measured length upon gel electrophoresis calibrated by coelectrophoresis of various DNA standard markers.
Differentially labeled: indicates that each extension product can be distinguished from all others because it has a different label attached and/or is of a different size and/or binds to a specifically labeled oligonucleotide. One of ordinary skill in the art will recognize that a variety of labels are available. Various factors affect the choice of the label. These include the effect of the label on the rate of hybridization and binding of the primer to the DNA, the sensitivity of the label, the ease of making the labeled primer, probe or extension products, the ability to automate, the available instrumentation, convenience, and the like.
Extension product: refers to the nucleotide sequence which is synthesized from the 3' end of the oligonucleotide primer and which is complementary to the strand to which the oligonucleotide primer is bound.
Flanking sequence: refers to the nucleotide sequence on either side of the part of the polymorphic region of the genetic locus to be amplified. "Unique flanking sequences" are those flanking sequences which are only found at one location within the genome.
Locus (or genetic locus): a specific position on a chromosome. Alleles of a locus are located at identical sites on homologous chromosomes.
Locus compatible marker: a DNA standard marker manufactured for use in gel electrophoretic determination of DNA fragment lengths of a polymorphic genetic locus from which it is derived through modification of the length of flanking sequence from a true or null allele of that locus. Locus compatible markers constitute part of the invention described herein.
Locus specific STR marker: a DNA standard marker manufactured for use in gel electrophoretic determination of DNA fragment lengths of a polymorphic genetic locus by subtracting or adding tandem repeat units to create novel alleles shorter than all known or common true alleles or longer than all known or common true alleles. Locus specific STR markers constitute another part of the invention described herein.
Locus specific primer: a primer that specifically hybridizes with a portion of a unique flanking sequence of a stated locus or its complementary strand, at least for one allele of the locus, and does not hybridize efficiently with other DNA sequences under the conditions used in the amplification method.
Multiplex polymerase chain reaction (mPCR): refers to a variation of PCR involving a procedure for simultaneously performing PCR on greater than two different sequences. In mPCR, after formation of two single stranded complimentary strands, a plurality of labeled paired oligonucleotide primers, where each pair is specific for a different sequence, are added. One primer of each pair is substantially complimentary for the part of the sequence in the sense strand and the other primer of each pair is substantially complimentary to a different part of the same sequence in the complementary antisense strand. The plurality of paired primers are annealed to their complementary sequence and simultaneously extended from each primer in each primer pair. The extension products then serve as a template for the other primer of each pair.
Polymorphism: refers to the genetic variation found in part of the genetic locus to be amplified.
Polymerase Chain Reaction (PCR): a technique in which cycles of denaturation, annealing with primer, and extension with DNA polymerase, are used to amplify the number of copies of a target DNA sequence by &gt;10.sup.6 times. The PCR process is described more fully in U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202, both of which are herein incorporated by reference.
Primers: two single-stranded oligonucleotides or DNA fragments which hybridize opposing strands of a locus such that the 3' termini of the primers are in closest proximity.
Primer pair: a set of primers including a 5' forward primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' reverse primer which hybridizes with the complement of the 3' end of the sequence to be amplified.
Primer site: the area of the target DNA to which a primer hybridizes.
Run time: the time required for a DNA fragment to migrate in an electric field through a fixed length of gel or other matrix and reach a detector.
Variant allele: a manufactured STR allele of a genetic locus incorporating repeat unit(s) or core repeat unit(s) of its polymorphism and either shorter or longer than all alleles or common alleles of its locus due to its variant number of repeat units. Variant alleles constitute part of the invention described herein.